NIGMS RO1, PI: Tao Lu, Ph.D. Title: Gene-specific responses to NF-?B through lysine and arginine methylation of p65 Abstract: Activation of the multi-functional transcription factor nuclear factor ?B (NF-?B), a central coordinator of immune responses, is tightly regulated in order to achieve its normal transient activation in response to stress. In many pathologies, NF-?B is activated abnormally, contributing to the development of a variety of disorders, including lung disease, chronic inflammatory diseases, cardiovascular disease, diabetes, and cancer. Thus, drugs that block NF-?B activation could be effective in treating these diseases. Understanding the molecular mechanism of NF-?B activation is the first step toward our long-term goal of identifying novel therapeutics. This proposal focuses on methylation as a novel mechanism ensuring precise control of NF-?B activity at its target genes. Recently, we discovered that lysine residues 218/221 (K218/221) and arginine residue 30 (R30) of the p65 subunit of NF-?B are methylated by histone-modifying enzymes. Our central hypothesis is that p65 R30 and K218/221 methylation differentially regulates NF-?B-dependent gene expression by affecting promoter binding, recruitment of transcriptional modifiers, and the physical properties of the NF-?B:DNA interaction. To test this central hypothesis, we will pursue two specific aims: Aim 1: Dissect the distinct impacts of p65 R30 and K218/221 methylation on the critical molecular events that lead to differential gene regulation. Aim 2: Determine the structural consequences of p65 R30 methylation, and discover the mechanisms of NF-?B-DNA sequence- specific effects on target gene promoters. Significance: The important findings from this study will identify the molecular mechanisms underlying p65 methylation-dependent gene-specific regulation, thus revealing how the extreme plasticity of biological responses is regulated by NF-?B, and offering deep insights into the development of NF-?B-associated diseases as well as innovative strategies for their therapeutic intervention.